Despite decades of intensive research, cancer remains the second leading cause of death in the United States. One in every two men and one in three women will develop cancer in their lifetime, with one in four men and one in five women dying from cancer. Though cancer has shown a slow decline since early 1990's, in part due to early detection, preventative measures, decreased tobacco use, advances in the field have done little to improve the survival outcome of patients with late-stage metastatic cancer. Standard care typically involves surgery, chemotherapy, and radiation, but these treatments often cause toxic side effects and may even promote cancer progression and metastasis (Sun, et al. (2012) Treatment-induced damage to the tumor microenvironment promotes prostate cancer therapy resistance through WNT16B. Nature medicine; Seyfried, et al. (2010) Does the existing standard of care increase glioblastoma energy metabolism? The lancet oncology 11: 811-813). While many primary tumors can be controlled with conventional therapies, these treatments are largely ineffective against long-term management of metastatic disease (Graeme, et al. (2004) The contribution of cytotoxic chemotherapy to 5-year survival in adult malignancies. Clinical Oncology 16).
Metastasis is a complex phenomenon in which cancer cells spread from a primary tumor to establish foci in a distal tissue. The specific changes which mediate metastasis remain unclear; however, the process generally involves local tumor growth, invasion through the basement membrane and surrounding tissue, intravasation into the blood vessels, dissemination and survival in circulation, extravasation from the vasculature, and re-establishment of tumors at distal tissues. As metastasis is responsible for over 90 percent of cancer-related deaths, there is a substantial need for novel treatments effective against metastatic cancer (Gupta & Massagué (2006) Cancer metastasis: building a framework. Cell 127: 679-695). While many primary tumors can be controlled with conventional therapies like surgery, chemotherapy, and radiation, these treatments are often ineffective against long-term management of metastatic disease which is responsible for 90 percent of cancer-related deaths (Graeme, et al. (2004) The contribution of cytotoxic chemotherapy to 5-year survival in adult malignancies. Clinical Oncology 16; Gupta & Massagué (2006) Cancer metastasis: building a framework. Cell 127: 679-695). There is a substantial need for novel treatments effective against metastatic cancer. The epithelial-to-mesenchymal transition (EMT) is the activation of a latent embryonic program causing a switch from epithelial to mesenchymal phenotype, and alterations in cell-cell/cell-matrix, which enhances cellular motility. Key cellular processes involved in EMT in vitro have been shown to affect metastatic spread in vivo, though metastasis is difficult to study in vivo due to the lack of adequate animal models.
Eighty-eight percent of ATP is made via oxidative phosphorylation in the mitochondria, through an oxygen-dependent pathway. Hypoxic conditions cause a shift to anaerobic fermentation, whereby ATP is produced through substrate level phosphorylation in an oxygen independent pathway. This adaptation to hypoxic mediated fermentation, which is an inefficient process for a rapidly dividing cell, requires HIF-1.
While the major oncogene and tumor suppressor gene mutations can be found in many different cancers, one of the only universal traits of tumor cells across tissue types is abnormal energy metabolism (Seyfried & Shelton (2010) Cancer as a metabolic disease. Nutrition & metabolism 7: 7). In the 1930s, Otto Warburg observed that cancer cells express abnormal energy metabolism characterized by very high rates of aerobic glycolysis (fermentation in the presence of oxygen) (Warburg (1956) On the origin of cancer cells. Science 123: 309-314; Warburg (1956) On respiratory impairment in cancer cells. Science 124: 269-270). This feature, known as The Warburg Effect, is a consequence of mitochondrial dysfunction and genetic mutations within the cancer cell (Seyfried & Shelton (2010) Cancer as a metabolic disease. Nutrition & metabolism 7: 7). While healthy cells derive the vast majority of their energy from ATP production by oxidative phosphorylation (OXPHOS) in the mitochondria, cancer cells rely almost exclusively on ATP production by substrate level phosphorylation (SLP) (Seyfried & Shelton (2010) Cancer as a metabolic disease. Nutrition & metabolism 7: 7). Nearly ubiquitously, cancers utilize SLP of glycolysis in the cytoplasm, as seen in FIG. 1, and, in some cancers, of glutaminolysis and the Kreb's Cycle (Lunt S Y, Vander Heiden M G (2011) Aerobic glycolysis: meeting the metabolic requirements of cell proliferation. Annual review of cell and developmental biology 27: 441-464; Medina (2001) Glutamine and cancer. The Journal of nutrition 131: 2539S-2542S; discussion 2550S-2531S). In fact, cancer cells undergo glycolysis at a rate up to 200-times that of healthy cells (Warburg (1956) On respiratory impairment in cancer cells. Science 124: 269-270). It is well documented that cancer cells across tissue types possess an array of mitochondrial damage, including loss of mitochondrial number, mitochondrial swelling, partial or total cristolysis, abnormalities in mitochondrial lipid composition, and absent, mutated, or decreased activity of mitochondrial enzymes involved in OXPHOS (Cuezva, et al. (2002) The bioenergetic signature of cancer: a marker of tumor progression. Cancer research 62: 6674-6681; Isidoro, et al. (2004) Alteration of the bioenergetic phenotype of mitochondria is a hallmark of breast, gastric, lung and oesophageal cancer. The Biochemical journal 378: 17-20; Arismendi-Morillo & Castellano-Ramirez (2008) Ultrastructural mitochondrial pathology in human astrocytic tumors: potentials implications pro-therapeutics strategies. Journal of electron microscopy 57: 33-42; Kiebish, et al. (2008) Cardiolipin and electron transport chain abnormalities in mouse brain tumor mitochondria: lipidomic evidence supporting the Warburg theory of cancer. Journal of lipid research 49: 2545-2601; Modica-Napolitano & Singh (2004) Mitochondrial dysfunction in cancer. Mitochondrion 4: 755-817; Kataoka, et al. (1991) Ultrastructural study of mitochondria in oncocytes. Ultrastructural pathology 15: 231-239). With this severe mitochondrial damage, cancer cells are unable to produce adequate amounts of ATP through OXPHOS to maintain viability and are forced to up-regulate SLP and glycolysis to survive (Seyfried & Shelton (2010) Cancer as a metabolic disease. Nutrition & metabolism 7: 7). Many of the genes that mediate this shift are known oncogenes and tumor suppressor genes. HIF-1α, IGF-1/PI3K/Akt, MYC, mTOR, and Ras upregulate glycolytic enzymes and GLUT transporter expression (Seyfried & Shelton (2010) Cancer as a metabolic disease. Nutrition & metabolism 7: 7; Miceli & Jazwinski (2005) Common and cell type-specific responses of human cells to mitochondrial dysfunction. Experimental Cell Research 302: 270-280); p53 and PTEN inhibit these responses and are thus inhibited (Liu & Feng (2012) PTEN, energy metabolism and tumor suppression. Acta biochimica et biophysica Sinica).
This fermentative phenotype causes cancers to excrete large quantities of lactate, creating an acidic tumor microenvironment that promotes epithelial to mesenchymal transition (EMT), invasion, and metastasis (Walenta, et al. (2000) High lactate levels predict likelihood of metastases, tumor recurrence, and restricted patient survival in human cervical cancers. Cancer research 60: 916-921; Dhup, et al. (2012) Multiple biological activities of lactic acid in cancer: influences on tumor growth, angiogenesis and metastasis. Current pharmaceutical design 18: 1319-1330). Lactate can also be returned to the cancer as glucose via the Cori Cycle, replenishing fuel for the glycolysis-dependent tumor cells, as seen in FIG. 1. Due to this metabolic deficiency, cancer cells have elevated rates of glucose consumption relative to healthy cells—a quality that underlies the use of fluorodeoxyglucose-PET scans as an important diagnostic tool for oncologists (Duranti, et al. (2012) PET scan contribution in chest tumor management: a systematic review for thoracic surgeons. Tumori 98: 175-184).
The Warburg Effect creates a glucose-dependency which can be targeted therapeutically (Seyfried & Shelton (2010) Cancer as a metabolic disease. Nutrition & metabolism 7: 7; Seyfried, et al. (2008) Targeting energy metabolism in brain cancer with calorically restricted ketogenic diets. Epilepsia 49 Suppl 8: 114-116). Ketogenic diets (KDs) are high fat, low or no carbohydrate diets that have been used to treat pediatric refractory epilepsy for decades (Katyal, et al. (2000) The ketogenic diet in refractory epilepsy: the experience of Children's Hospital of Pittsburgh. Clinical pediatrics 39: 153-159). KDs naturally suppress appetite and often lead to dietary energy restriction (DER) and body weight loss (Katyal, et al. (2000) The ketogenic diet in refractory epilepsy: the experience of Children's Hospital of Pittsburgh. Clinical pediatrics 39: 153-159) or decreased lean body mass (Katyal, et al. (2000) The ketogenic diet in refractory epilepsy: the experience of Children's Hospital of Pittsburgh. Clinical pediatrics 39: 153-159; Paoli, et al. (2012) Ketogenic diet does not affect strength performance in elite artistic gymnasts. Journal of the International Society of Sports Nutrition 9: 34; Johnstone, et al. (2008) Effects of a high-protein ketogenic diet on hunger, appetite, and weight loss in obese men feeding ad libitum. The American journal of clinical nutrition 87: 44-55; Hussain, et al. (2012) Effect of low-calorie versus low-carbohydrate ketogenic diet in type 2 diabetes. Nutrition 28: 1016-1021; Volek, et al. (2004) Comparison of energy-restricted very low-carbohydrate and low-fat diets on weight loss and body composition in overweight men and women. Nutrition & metabolism 1: 13). While low carbohydrate or KDs promote weight loss in overweight individuals, they are known to spare muscle wasting during DER (Paoli, et al. (2012) Ketogenic diet does not affect strength performance in elite artistic gymnasts. Journal of the International Society of Sports Nutrition 9: 34; Manninen (2006) Very-low-carbohydrate diets and preservation of muscle mass. Nutrition & metabolism 3: 9; Cahill (2006) Fuel metabolism in starvation. Annual review of nutrition 26: 1-22; Veech (2004) The therapeutic implications of ketone bodies: the effects of ketone bodies in pathological conditions: ketosis, ketogenic diet, redox states, insulin resistance, and mitochondrial metabolism. Prostaglandins, leukotrienes, and essential fatty acids 70: 309-319). DER has been shown to slow disease progression in a variety of cancers, including brain, prostate, mammary, pancreas, lung, gastric, and colon (Seyfried & Shelton (2010) Cancer as a metabolic disease. Nutrition & metabolism 7: 7; Zuccoli, et al. (2010) Metabolic management of glioblastoma multiforme using standard therapy together with a restricted ketogenic diet: Case Report. Nutrition & metabolism 7: 33; Mavropoulos, et al. (2006) Is there a role for a low-carbohydrate ketogenic diet in the management of prostate cancer? Urology 68: 15-18; Zhou, et al. (2007) The calorically restricted ketogenic diet, an effective alternative therapy for malignant brain cancer. Nutrition & metabolism 4: 5; Mavropoulos, et al. (2009) The effects of varying dietary carbohydrate and fat content on survival in a murine LNCaP prostate cancer xenograft model. Cancer prevention research (Philadelphia, Pa.) 2: 557-565; Otto, et al. (2008) Growth of human gastric cancer cells in nude mice is delayed by a ketogenic diet supplemented with omega-3 fatty acids and medium-chain triglycerides. BMC cancer 8: 122; Masko, et al. (2010) Low-carbohydrate diets and prostate cancer: how low is “low enough”? Cancer prevention research (Philadelphia, Pa.) 3: 1124-1131; Tisdale & Brennan; A comparison of long-chain triglycerides and medium-chain triglycerides on weight loss and tumor size in a cachexia model.pdf; Wheatley, et al. (2008) Low-carbohydrate diet versus caloric restriction: effects on weight loss, hormones, and colon tumor growth in obese mice. Nutrition and cancer 60: 61-68; Rossifanelli, et al. (1991) Effect of Energy Substrate Manipulation on Tumor-Cell Proliferation in Parenterally Fed Cancer-Patients. Clinical Nutrition 10: 228-232). DER appears to facilitate its anti-cancer effects through several metabolic pathways, including inhibition of the IGF-1/PI3K/Akt signaling pathway which promotes proliferation and angiogenesis and inhibits apoptosis (Mukherjee, et al. (2002) Dietary restriction reduces angiogenesis and growth in an orthotopic mouse brain tumour model. British journal of cancer 86: 1615-1621; Mukherjee, et al. (1999) Energy intake and prostate tumor growth, angiogenesis, and vascular endothelial growth factor expression. Journal of the National Cancer Institute 91: 512-523; Thompson, et al. (2004) Effect of dietary energy restriction on vascular density during mammary carcinogenesis. Cancer research 64: 5643-5650; Hursting, et al. (2010) Calories and carcinogenesis: lessons learned from 30 years of calorie restriction research. Carcinogenesis 31: 83-89; Thompson, et al. (2003) Dietary energy restriction in breast cancer prevention. Journal of mammary gland biology and neoplasia 8: 133-142; Thompson, et al. (2004) Identification of the apoptosis activation cascade induced in mammary carcinomas by energy restriction. Cancer research 64: 1541-1545; Zhu, et al. (2005) Effects of dietary energy repletion and IGF-1 infusion on the inhibition of mammary carcinogenesis by dietary energy restriction. Molecular carcinogenesis 42: 170-176). DER has been shown to induce apoptosis in astrocytoma cells but protect normal brain cells from death through activation of adenosine monophosphate kinase (AMPK) (Mukherjee, et al. (2008) Differential effects of energy stress on AMPK phosphorylation and apoptosis in experimental brain tumor and normal brain. Molecular cancer 7: 37). The KD has been successfully used as an adjuvant therapy for Glioblastoma Multiforme (GBM) in a small number of case reports with patients exhibiting marked improvements in quality of life, dramatic slowing of tumor growth, or disappearance of tumor altogether (Zuccoli, et al. (2010) Metabolic management of glioblastoma multiforme using standard therapy together with a restricted ketogenic diet: Case Report. Nutrition & metabolism 7: 33; Nebeling & Lerner (1995) Implementing a ketogenic diet based on medium-chain triglyceride oil in pediatric patients with cancer. Journal of the American Dietetic Association 95: 693-697). Furthermore, a pilot trial of patients with advanced metastatic disease of varying tissue types reported that the KD improved emotional functioning and quality of life in terminally ill patients (Schmidt, et al. (2011) Effects of a ketogenic diet on the quality of life in 16 patients with advanced cancer: A pilot trial. Nutr Metab (Lond). 2011 Jul. 27; 8(1):54).
As such, any conditions which restrict glucose availability (or impair glycolysis) while providing alternative energy sources for healthy cells, can selectively starve cancer cells while leaving normal cells unharmed. Metabolic therapy in the forms of dietary energy restriction or the ketogenic diet (KD) have been shown to elicit anti-cancer effects in a variety of cancers, likely by restricting glucose availability to the tumor and by inhibiting oncogenes that promote cancer progression (Seyfried & Shelton (2010) Cancer as a metabolic disease. Nutrition & metabolism 7: 7; Zhou, et al. (2007) The calorically restricted ketogenic diet, an effective alternative therapy for malignant brain cancer. Nutrition & metabolism 4: 5; Zuccoli, et al. (2010) Metabolic management of glioblastoma multiforme using standard therapy together with a restricted ketogenic diet: Case Report. Nutrition & metabolism 7: 33; Mavropoulos, et al. (2006) Is there a role for a low-carbohydrate ketogenic diet in the management of prostate cancer? Urology 68: 15-18). The two most abundant and physiologically relevant ketone bodies are acetoacetate (ACA) and β-hydroxybutyrate (βHB). Ketone bodies are metabolized exclusively in the mitochondria via the Kreb's Cycle and OXPHOS coupled to the electron transport chain. These metabolic strategies elevate blood ketone concentrations. Due to mitochondrial damage, most cancers are unable to utilize ketones for energy (Maurer, et al. (2011) Differential utilization of ketone bodies by neurons and glioma cell lines: a rationale for ketogenic diet as experimental glioma therapy. BMC Cancer. 2011 Jul. 26; 11:315; Cuezva, et al. (2002) The bioenergetic signature of cancer: a marker of tumor progression. Cancer research 62: 6674-6681; Fearon, et al. (1988) Cancer cachexia: influence of systemic ketosis on substrate levels and nitrogen metabolism. The American journal of clinical nutrition 47: 42-48; Sawai, et al. (2004) Growth-inhibitory effects of the ketone body, monoacetoacetin, on human gastric cancer cells with succinyl-CoA: 3-oxoacid CoA-transferase (SCOT) deficiency. Anticancer research 24: 2213-2217; Seyfried, et al. (2003) Role of glucose and ketone bodies in the metabolic control of experimental brain cancer. British journal of cancer 89: 1375-1457; Oudard, et al. (1997) Gliomas are driven by glycolysis: putative roles of hexokinase, oxidative phosphorylation and mitochondrial ultrastructure. Anticancer research 17: 1903-1911; John (2001) Dysfunctional mitochondria, not oxygen insufficiency, cause cancer cells to produce inordinate amounts of lactic acid: the impact of this on the treatment of cancer. Medical hypotheses 57: 429-460; Wu, et al. (2007) Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells. American journal of physiology Cell physiology 292: C125-136; Skinner, et al. (2009) Ketone bodies inhibit the viability of human neuroblastoma cells. Journal of pediatric surgery 44: 212; Sawai, et al. (2004) Growth-inhibitory effects of the ketone body, monoacetoacetin, on human gastric cancer cells with succinyl-CoA: 3-oxoacid CoA-transferase (SCOT) deficiency. Anticancer research 24: 2213-2217; Tisdale & Brennan (1983) Loss of acetoacetate coenzyme A transferase activity in tumours of peripheral tissues. British journal of cancer 47: 293-297).
Furthermore, ketones have been shown to inhibit cancer cell proliferation (Skinner, et al. (2009) Ketone bodies inhibit the viability of human neuroblastoma cells. Journal of pediatric surgery 44: 212; Sawai, et al. (2004) Growth-inhibitory effects of the ketone body, monoacetoacetin, on human gastric cancer cells with succinyl-CoA: 3-oxoacid CoA-transferase (SCOT) deficiency. Anticancer research 24: 2213-2217; Magee, et al. (1979) The inhibition of malignant cell growth by ketone bodies. The Australian journal of experimental biology and medical science 57: 529-539). Many cancers do not express the Succinyl-CoA: 3-ketoacid CoA-Transferase (SCOT) enzyme, which is required for ketone body metabolism (Sawai, et al. (2004) Growth-inhibitory effects of the ketone body, monoacetoacetin, on human gastric cancer cells with succinyl-CoA: 3-oxoacid CoA-transferase (SCOT) deficiency. Anticancer research 24: 2213-2217; Tisdale & Brennan (1983) Loss of acetoacetate coenzyme A transferase activity in tumours of peripheral tissues. British journal of cancer 47: 293-297). βHB administration rescues healthy brain cells from glucose withdrawal-induced cell death but does not protect glioma cells (Maurer, et al. (2011) Differential utilization of ketone bodies by neurons and glioma cell lines: a rationale for ketogenic diet as experimental glioma therapy. BMC Cancer. 2011 Jul. 26; 11:315). While ketones are not an energy source for cancer cells, they are an efficient energy substrate for healthy tissue in the rest of the body. Ketones have been shown to inhibit cancer cell growth and proliferation in vitro in a variety of cell lines, including gastric cancer, transformed lymphoblasts, kidney cancer, HeLa cells, and melanoma (Magee, et al. (1979) The inhibition of malignant cell growth by ketone bodies. The Australian journal of experimental biology and medical science 57: 529-539; Sawai, et al. (2004) Growth-inhibitory effects of the ketone body, monoacetoacetin, on human gastric cancer cells with succinyl-CoA: 3-oxoacid CoA-transferase (SCOT) deficiency. Anticancer research 24: 2213-2217). It is unclear exactly how ketones elicit their anti-cancer effects. Ketone bodies are known to inhibit glycolysis, which may contribute to their efficacy (Wu & Thompson (1988) The effect of ketone bodies on alanine and glutamine metabolism in isolated skeletal muscle from the fasted chick. The Biochemical journal 255: 139-144). Additionally, ketones are transported into the cell via the monocarboxylate transporters (MCTs) which are also responsible for exporting the fermentation product lactate from the cell into the circulation. Lactate confers an acidic tumor microenvironment and is known to play a large role in invasion and metastasis (Dhup, et al. (2012) Multiple biological activities of lactic acid in cancer: influences on tumor growth, angiogenesis and metastasis. Current pharmaceutical design 18: 1319-1330). Furthermore, it has been well-documented that both calorie restriction and fasting, conditions where ketones take over as a primary fuel, possess very potent anti-cancer effects, further supporting the observation that cancer cells cannot thrive by using ketone bodies for fuel (Hursting, et al. (2010) Calories and carcinogenesis: lessons learned from 30 years of calorie restriction research. Carcinogenesis 31: 83-89; Lee, et al. (2012) Starvation, detoxification, and multidrug resistance in cancer therapy. Drug resistance updates: reviews and commentaries in antimicrobial and anticancer chemotherapy 15: 114-122).
However, the present methods only provide enhanced anticancer effects. As such, enhanced anticancer therapies are required which reduce cancer growth as well as metastasis.